Enhanced purification of antibodies and antibody fragments by apatite chromatography

ABSTRACT

Methods are disclosed for use of apatite chromatography, particularly without reliance upon phosphate gradients, for purification or separation of at least one intact non-aggregated antibody, or at least one immunoreactive antibody fragment, from an impure preparation. Integration of such methods into multi-step procedures with other fractionation methods are additionally disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.Nos. 61/011,513 filed Jan. 18, 2008; 61/062,663 filed Jan. 28, 2008;61/069,859 filed Mar. 19, 2008; 61/070,841 filed Mar. 27, 2008;61/135,787 filed Jul. 24, 2008; 61/189,467 filed Aug. 20, 2008, each ofwhich are expressly incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates in certain embodiments to methods for enhancingpurification of antibodies and immunoreactive antibody fragments byapatite chromatography in the presence of one or more of boratecompounds, sulfate compounds, monocarboxylate compounds, and/or in thepresence of calcium compounds. In certain embodiments, the invention maypermit more effective separation of intact non-aggregated antibody fromunwanted fragments, aggregated antibody, and other contaminants. Inother embodiments, the invention may permit more effective purificationof immunoreactive antibody fragments. In these or other embodiments, theinvention may improve pH control during the separation.

BACKGROUND OF THE INVENTION

Hydroxyapatite [HA] is a crystalline mineral of calcium phosphate with astructural formula of Ca₁₀(PO₄)₆(OH)₂. Fluorapatite may be prepared byfluoridating hydroxyapatite, creating a mineral with the structuralformula Ca₁₀(PO₄)₆F₂. Protein-reactive sites on both minerals includepairs of positively charged calcium ions (C-sites) and triplets ofnegatively charged phosphate groups (P-sites). C-sites interact withproteins via HA calcium chelation by protein carboxyl clusters. C-sitesinteract with phosphorylated solutes such as DNA, endotoxin,phosphoproteins, and lipid enveloped viruses via HA calcium coordinationby solute phosphate residues. Calcium chelation and coordination aresometimes referred to as calcium affinity. P-sites interact withproteins via phosphoryl cation exchange with positively charged proteinamino acid residues (Gorbunoff, Analytical Biochemistry 136 425 (1984);Kawasaki, J Chromatography 152 361 (1985)). Hydroxyapatite is mostcommonly eluted with phosphate gradients. The strong calcium affinity ofphosphate suspends calcium chelation and coordination interactions,while its ionic character suspends phosphoryl cation exchangeinteractions. Some applications elute hydroxyapatite with combinationsof phosphate and chloride salts. Chlorides preferentially elute thephosphoryl cation exchange interaction while having relatively littleeffect on calcium affinity interactions. (Gagnon et al, BioprocessInternational, 4(2) 50 (2006)).

Native hydroxyapatite and fluorapatite can be converted tocalcium-derivatized forms by exposure to soluble calcium in the absenceof phosphate. (Gorbunoff, Anal. Biochem., 136 425 (1984)). This convertsP-sites into secondary C-sites, abolishing phosphoryl cation exchangeinteractions, increasing the number of C-sites, and fundamentallyaltering the selectivity of the apatite support. Small alkaline proteinstypified by lysozyme (13.7-14.7 Kda, pI 10.7) and ribonuclease (14.7kDa, pI 9.5-9.8) fail to bind to calcium-derivatized apatites, but mostother proteins bind so strongly that even 3 M calcium chloride isinadequate to achieve elution (Gorbunoff). Other chloride salts alsofail to achieve elution. Calcium-derivatized apatites are restored totheir native forms by exposure to phosphate buffer, at which point theymay be eluted by methods commonly applied for elution of native apatitesupports.

The effects of different salts on the selectivity of a given apatite areunpredictable. For example, in the absence of phosphate, sodium chlorideis unable to elute most IgG monoclonal antibodies from nativehydroxyapatite, even at concentrations in excess of 4 moles per liter(Gagnon et al, 2006, Bioprocess International, 4(2) 50). This impliesextremely strong binding. In exclusively phosphate gradients, IgG istypically one of the latest eluting proteins, usually requiring 100-150mM phosphate. This also implies strong binding. When eluted with acombination of lower concentrations of both salts, such as 0.25 M sodiumchloride and 50 mM phosphate however, IgG is one of the earliest elutingproteins. Other paradoxes reinforce the point: increasing the sodiumchloride concentration in the presence of phosphate, which causes IgG tobind less strongly, has the opposite effect on DNA (Gagnon et al, 2005,Bioprocess International, 3(7) 52-55). Additionally, lysozyme elutes ata higher phosphate concentration than BSA in the absence of sodiumchloride but fails to bind in the presence of 1 M sodium chloride.

Ammonium sulfate, sodium sulfate, and other sulfate salts are commonlyused for precipitation of proteins, or to cause proteins to bind tohydrophobic interaction chromatography media. They can also be used toenhance binding with biological affinity chromatography media such asprotein A, and have even been reported to cause proteins to bind to ionexchangers (Gagnon, 1996, Purification Tools for Monoclonal Antibodies,ISBN 0-9653515-9-9; Mevarech et al, 1976, Biochemistry 15, 2383-2387;Leicht et al, 1981, Anal. Biochem., 114, 186-192; Arakawa et al, 2007,J. Biochem, Biophys. Met., 70, 493-498). Sulfates have occasionally beenreported for elution of ion exchangers at low concentrations forresearch applications but are seldom exploited in preparativeapplications due to concerns over protein precipitation (Kopaciewicz etal, 1983, J. Chromatogr., 266 3-21; Gooding et al, 1984, J. Chromatogr.,296, 321-328; Rounds et al, 1984, J. Chromatogr., 283 37-45). None ofthese methods is an appropriate model for apatites because none of themexploits calcium affinity for binding.

Several authors have concluded that, “The presence of . . . (NH₄)₂SO₄seems not to affect the elution [of hydroxyapatite].” (Karlsson et al,1989, in Protein Purification: Principles, High Resolution Methods, andApplications, Chapter 4, ISBN 0-89573-122-3). Even this referencementions the application of sulfate strictly in the context of phosphategradients. In the rare cases where alternatives to phosphate as aprimary eluting salt have been discussed in the literature, suggestionshave included calcium chloride, citrate and fluoride salts, but withoutmention of sulfates (Gagnon, 1996; Karlsson et al, 1989; Gorbunoff).Other publications indicate that sulfate salts in particular should beunsuitable as primary eluting agents for hydroxyapatite because “ . . .SO₃H do[es] not form complexes with calcium.” (Gorbunoff).

Borate salts have been likewise overlooked. Borate is occasionally usedin the field of chromatography as a buffering agent at pH values fromabout 8.8 to 9.8 (pK ˜9.24). It is also used infrequently at alkaline pHto modify the charge characteristics of cis-diol compounds toselectively enhance their retention on anion exchangers. In contrast tophosphates, chlorides, and sulfates, all of which exhibit molarconductivities of about 90 mS/cm, a 1 M solution of borate at pH 7 has amolar conductivity of about 9 mS.

Acetates have been compared to chlorides for hydroxyapatite separationof IgG from aggregates and were found to support inferior fractionation(Gagnon et al, Practical issues in the industrial use of hydroxyapatitefor purification of monoclonal antibodies, Poster, 22^(nd) nationalmeeting of the American Chemical Society, San Francisco, Sep. 10-14,2006<http://www.validated.com/revalbio/pdffiles/ACS_CHT_(—)02.pdf>.Monocarboxylic acid salts have been neglected, and the elution potentialof monocarboxylic zwitterions totally so.

Hydroxyapatite is used for purification of antibodies and antibodyfragments (Bowles et al, Int. J. Pharmacol., 10 537 (1988); Guerrier etal, J. Chromatography B, 755 37 (2001); Gagnon et al, BioProcess Int.,4(2) 50 (2006)). The column is usually equilibrated and the sampleapplied in a buffer that contains a low concentration of phosphate.Adsorbed antibodies are usually eluted in an increasing gradient ofphosphate salts. Alternatively, they may be eluted in an increasinggradient of chloride salts but both elution formats impose disadvantageson purification procedures. The high phosphate concentration in whichantibodies elute in phosphate gradients has strong buffer capacity thatmay interfere with subsequent purification steps. The high conductivityat which antibodies elute in chloride gradients may also interfere withdownstream steps. Both situations require either that the elutedantibody be diluted extensively, or that it be buffer-exchanged, forexample by diafiltration, in order to modify the conditions to renderthe antibody preparation suitable for application to a subsequentpurification step. Dilution and buffer exchange have a negative impacton process economics. As a result, apatite chromatography steps areoften placed at the end of a purification process. This tends toeliminate them from consideration as capture steps. It also discouragesthe use of HA as an intermediate step. A further disadvantage ofchloride gradients is that the application of chloride to hydroxyapatitecauses an uncontrolled reduction of pH. Acidic pH causes destruction ofhydroxyapatite and risks adverse affects to antibodies bound to it.

Another limitation of hydroxyapatite with antibody purification is thatIgG binding capacity is reduced at elevated conductivity values. Thisstrongly reduces its versatility since the salt concentration of cellculture supernatants and antibody-containing fractions from purificationmethods such as ion exchange and hydrophobic interaction chromatography,confers sufficient conductivity to reduce the IgG binding capacity ofhydroxyapatite to such an extent that it may not be useful for aparticular application. This disadvantage can be overcome bydiafiltration or dilution of the sample prior to its application to thehydroxyapatite column, but as noted above, these operations increase theexpense of the overall purification process. Alternatively, thedisadvantage can be ameliorated by using a larger volume ofhydroxyapatite, but this increases process expense by requiring largercolumns and larger buffer volumes. It also causes the antibody to elutein a larger volume of buffer, which increases overall process time inthe subsequent purification step.

SUMMARY OF THE INVENTION

The present invention in certain embodiments relates to methods offractionating or purifying a desired antibody or immunoreactive antibodyfragment from an impure preparation by contacting said preparation witha native or calcium-derivatized apatite chromatography support, theneluting the support in the presence of an ionic species which is asulfate, borate, monocarboxylic organic acid salt or monocarboxyliczwitterion. In certain embodiments the ionic species is the primaryeluting ion in the eluent. In certain embodiments the eluent issubstantially free of phosphate as an eluting ion.

In certain embodiments of the inventions, a method for purifying anantibody or antibody fragment from an impure preparation is providedwherein the impure preparation is contacted with an apatitechromatography support in either the calcium derivatized form or in itsnative form and the apatite support is converted to the other form priorto elution of the antibody or antibody fragment.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of some embodiments of the invention include thefollowing: 1) Calcium-derivatized apatites support higher bindingcapacity than native hydroxyapatite for most antibodies, even at highconductivity values, thereby making apatite chromatography moreeffective as a capture method, or as an intermediate fractionation stepfollowing high-salt elution from another fractionation step such as ionexchange or hydrophobic interaction chromatography; 2)Calcium-derivatized apatites also produce unique selectivities that mayenable effective antibody or antibody fragment fractionation, includingremoval of aggregates, in situations where native apatites fail to doso; 3) Antibodies or fragments thereof may be bound to a native apatitesupport which is then converted to the calcium-derivatized form toachieve a particular selectivity for elution or; 4) Antibodies orfragments thereof may be bound to a calcium-derivatized apatite supportwhich is them converted to the native form for elution. 5) Sulfate,borate, and certain monocarboxylic acids or zwitterions are able toelute antibodies from apatite supports in the absence of phosphate; 6)Elution in the presence of sulfate, borate, or monocarboxylic acids orzwitterions produces unique selectivities that permit effectivefractionation of antibodies or antibody fragments, including removal ofaggregates, that may not be adequately served by elution with phosphateor by combinations of phosphate and chloride; 7) Borate permits elutionof antibodies and antibody fragments at low conductivity values, anddoes so without imposing significant buffer capacity at neutral pH,thereby facilitating use of the eluted antibody or antibody fragment insubsequent ion exchange chromatography steps without the necessity forintervening steps such as diafiltration; 8) Borate and certainmonocarboxylic acids or zwitterions create an increase in pH on contactwith apatites which can be used to counteract the effect of chlorides onpH, thereby attenuating or eliminating the pH reduction that otherwiseaccompanies the introduction of chlorides; 9) Sulfate enhances theseparation of antibodies and antibody fragments from phosphorylatedcontaminants.

In certain embodiments the ionic species is borate. In certainembodiments the borate is sodium borate or potassium borate. In certainsuch embodiments the primary eluting ion is borate. In certainembodiments the borate is present at a pH where the borate lackssubstantial buffering capacity; in certain such embodiments the pH isless than 8.7. In certain other embodiments the borate is present atgreater than 50 mM and at a pH where the borate has substantialbuffering capacity; in certain such embodiments the pH is 8.7 orgreater.

In certain embodiments the ionic species is sulfate. In certainembodiments the sulfate is sodium or potassium sulfate. In certainembodiments the sulfate is the primary eluting ion.

In certain embodiments the ionic species is a monocarboxylic acid salt.In certain such embodiments the monocarboxylate acid anion is formate,acetate, lactate, succinate, pyruvate, gluconate, glucuronate orproprionate. In certain embodiments the monocarboxylate is the primaryeluting ion.

In still other embodiments the ionic species is a monocarboxyliczwitterion. In certain such embodiments the monocarboxylate zwitterionis glycine, proline, lysine or histidine.

In some embodiments, the impure antibody or fragment preparation may beapplied to the apatite chromatography support under conditions thatpermit the binding of protein contaminants, intact non-aggregatedantibody, antibody fragments, and aggregated antibody, with purificationbeing achieved subsequently by application of an elution gradient. Thismode of chromatography is often referred to as bind-elute mode.

In some embodiments, the impure antibody or fragment preparation may beapplied to the apatite chromatography support under conditions thatprevent the binding of intact non-aggregated antibody or the desiredantibody fragment while binding contaminants. This mode of applicationis often referred to as flow-though mode. Bound contaminants may beremoved subsequently from the column by means of a cleaning step.

Suitable apatite chromatography supports include native hydroxyapatite,calcium-derivatized hydroxyapatite, native fluorapatite, andcalcium-derivatized fluorapatite.

In certain embodiments, elution may be achieved exclusively by means ofincreasing the concentration of the ionic species such as borate,sulfate, or monocarboxylic acids or zwitterions. In certain of suchembodiments such elution is achieved with a single ionic species as theeluting ion, e.g., borate or sulfate.

In some embodiments, elution may be achieved by borate in combinationwith calcium, magnesium, phosphate, sulfate, chloride, monocarboxylicacids or zwitterions, arginine, glycine, urea, or nonionic organicpolymers.

In some embodiments, elution may be achieved by sulfate in combinationwith calcium, magnesium, phosphate, borate, chloride, monocarboxylicacids or zwitterions, arginine, glycine, urea, or nonionic organicpolymers.

In some embodiments, elution may be achieved by monocarboxylic acids orzwitterions in combination with calcium, magnesium, phosphate, borate,sulfate, chloride, arginine, glycine, urea, or nonionic organicpolymers.

In certain embodiments, the method for purifying an antibody orimmunoreactive antibody fragment from an impure preparation containingsaid antibody or antibody fragment includes the steps of (a) contactingthe impure preparation with an apatite chromatography support, whereinthe apatite chromatography support is in a calcium-derivatized form whenit is contacted with the antibody or antibody fragment and (b)substantially converting the calcium-derivatized apatite chromatographysupport to its native form prior to eluting the antibody or antibodyfragment. In certain such embodiments the antibody or antibody fragmentis eluted with phosphate as the primary eluting ion.

In certain embodiments, the method for purifying a non-aggregatedantibody or immunoreactive antibody fragment from an impure preparationcontaining said antibody or antibody fragment involves the steps of (a)contacting the impure preparation with an apatite chromatographysupport, wherein the apatite chromatography support is in its nativeform when it is contacted with the antibody or antibody fragment and (b)substantially converting the native form apatite chromatography supportto a calcium-derivatized form prior to eluting the antibody or antibodyfragment. In certain such embodiments the conversion of the apatitechromatography support to the calcium derivatized form causes elution ofthe antibody or immunoreactive fragment of interest.

In certain embodiments, the antibody or antibody fragment is a mammalianimmunoglobulin of the class IgA, IgD, IgE, IgG, or IgM of monoclonal orpolyclonal origin, an avian immunoglobulin of the class IgY, or a fusionprotein containing a portion of an antibody.

In certain embodiments, the antibody fragment is Fab, F(ab′)₂, Fv, scFv,Fd, mAb, dAb or another fragmentary composition that retainantigen-binding function.

Embodiments of the invention may be practiced in combination with one ormore other purification methods, including but not limited to sizeexclusion chromatography, protein A and other forms of affinitychromatography, anion exchange chromatography, cation exchangechromatography, hydrophobic interaction chromatography, mixed modechromatography, and various filtration methods. It is within the abilityof a person of ordinary skill in the art to develop appropriateconditions for these methods and integrate them with the inventionherein to achieve purification of a particular antibody or antibodyfragment.

Terms are defined so that the invention may be understood more readily.Additional definitions are set forth throughout this disclosure.

“Apatite chromatography support” refers to a mineral of calcium andphosphate in a physical form suitable for the performance ofchromatography. Examples include but are not limited to hydroxyapatiteand fluorapatite. This definition is understood to include both thenative and calcium-derivatized forms of an apatite chromatographysupport.

“Salt” refers to an aqueous-soluble ionic compound formed by thecombination of negatively charged anions and positively charged cations.The anion or cation may be of organic or inorganic origin. Anions oforganic origin include but are not limited to acetate, lactate, malate,and succinate. Anions of inorganic origin include but are not limited tochloride, borate, sulfate, and phosphate. Cations of organic origininclude but are not limited to arginine and lysine. Cations of inorganicorigin include but are not limited to sodium, potassium, calcium,magnesium, and iron.

“Borate” refers to ionic compounds of boron and oxygen such as, but notlimited to boric acid, sodium borate, and potassium borate.

“Phosphate” refers to salts based on phosphorus (V) oxoacids such as,but not limited to, sodium phosphate and potassium phosphate.

“Sulfate” refers to salts based on sulfur (VI) oxoacids such as, but notlimited to sodium sulfate and ammonium sulfate.

“Chloride” refers to salts such as, but not limited to sodium chlorideand potassium chloride.

“Monocarboxylic acid salt” or “Monocarboxylate” refers to organic acidsalts having a single carboxylic acid moiety including but not limitedto the sodium or potassium salts of formic, acetic, propionic, lactic,pyruvic, gluconic, or glucuronic acid.

“Monocarboxylic zwitterion” refers to a molecule containing a singlecarboxyl moiety and at least one moiety with a positive charge. Suitableexamples include but are not limited to the amino acids glycine,proline, lysine, and histidine.

“Nonionic organic polymer” refers to any uncharged linear or branchedpolymer of organic composition. Examples include, but are not limitedto, dextrans, starches, celluloses, polyvinylpyrrolidones, polypropyleneglycols, and polyethylene glycols of various molecular weights.Polyethylene glycol has a structural formula HO—(CH₂—CH₂—O)_(n)—H.Examples include, but are not limited to, compositions with an averagepolymer molecular weight ranging from 100 to 10,000 daltons. The averagemolecular weight of commercial PEG preparations is typically indicatedby a hyphenated suffix. For example, PEG-600 refers to a preparationwith an average molecular weight of about 600 daltons.

“Buffering compound” refers to a chemical compound employed for thepurpose of stabilizing the pH of an aqueous solution within a specifiedrange. Phosphate is one example of a buffering compound. Other commonexamples include but are not limited to compounds such as acetate,morpholinoethanesulfonic acid (MES), Tris-hydroxyaminomethane (Tris),and hydroxyethylpiperazinesulfonic acid (HEPES).

“Buffer” refers to an aqueous formulation comprising a bufferingcompound and other components required to establish a specified set ofconditions to mediate control of a chromatography support. The term“equilibration buffer” refers to a buffer formulated to create theinitial operating conditions for a chromatographic operation. “Washbuffer” refers to a buffer formulated to displace unbound contaminantsfrom a chromatography support. “Elution buffer” refers to a bufferformulated to displace the one or more biomolecules from thechromatography support.

“Biomolecule” refers to a molecule of biological origin, composite, orfragmentary form thereof. Examples include but are not limited toproteins, polynucleotides, endotoxins, and viruses. Examples of proteinsinclude but are not limited to antibodies, enzymes, growth regulators,clotting factors, and phosphoproteins. Examples of polynucleotidesinclude DNA and RNA. Examples of viruses include enveloped andnon-enveloped viruses.

“Antibody” refers to any immunoglobulin or composite form thereof. Theterm may include, but is not limited to polyclonal or monoclonalantibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived fromhuman or other mammalian cell lines, including natural or geneticallymodified forms such as humanized, human, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, grafted, and in vitro generatedantibodies. “Antibodies” may also include composite forms including butnot limited to fusion proteins containing an immunoglobulin moiety.

“Antibody fragment” refers to any antibody fragment such as Fab,F(ab′)₂, Fv, scFv, Fd, mAb, dAb or other compositions that retainantigen-binding function. Antibody fragments may be derived from humanor other mammalian cell lines, including natural or genetically modifiedforms such as humanized, human, chimeric, synthetic, recombinant,hybrid, mutated, grafted, and in vitro generated, from sources includingbut not limited to bacterial cell lines, insect cell lines, plant celllines, yeast cell lines, or cell lines of other origin. Antibodyfragments may also be derived by controlled lysis of purified antibodywith enzymes such as, but not limited to ficin, papain, or pepsin.

“Antibody aggregate” refers to an association of at least twoantibodies. The association may be either covalent or non-covalentwithout respect to the mechanism by which the antibodies are associated.The association may be direct between the antibodies or indirect throughother molecules that link the antibodies together. Examples of thelatter include but are not limited to disulfide linkages with otherproteins, hydrophobic associations with lipids, charge associations withDNA, affinity associations with leached protein A, or mixed modeassociations with multiple components.

“Non-antibody proteins” refers to proteins formulated into antibodyproduction media and/or to proteins produced by the cell line or hostduring antibody production.

“Antibody preparation” refers to any composition containing an intactnon-aggregated antibody. Said preparation may contain antibody fragmentsand/or aggregates. Non-antibody proteins and other contaminants,potentially including but not limited to nucleic acids, endotoxin, andvirus may also be present. An impure preparation from which an antibodyis to be purified according to the invention can be an antibodypreparation.

“Antibody fragment preparation” refers to any composition containing animmunoreactive antibody fragment. Said preparation may contain intactantibodies and/or aggregates. Non-antibody proteins and othercontaminants, potentially including but not limited to host cellproteins, nucleic acids, endotoxin, and virus may also be present. Animpure preparation from which an antibody fragment is to be purifiedaccording to the invention can be an antibody preparation.

As it relates to the invention herein, the term “bind-elute mode” refersto an operational approach to chromatography in which the bufferconditions are established so that the intact non-aggregated antibody orantibody fragment, and contaminants, bind to the column uponapplication, with fractionation being achieved subsequently bymodification of the buffer conditions.

As it relates to the invention herein, the term “flow-through mode”refers to an operational approach to chromatography in which the bufferconditions are established so that the intact non-aggregated antibody orantibody fragment flows through the column upon application whilecontaminants are selectively retained, thus achieving their removal.

“Preparative applications” refers to situations in which the inventionis practiced for the purpose of purifying intact non-aggregated antibodyfor research, diagnostic, or therapeutic applications. Such applicationsmay be practiced at any scale, ranging from milligrams to kilograms ofantibody per batch.

Materials

1. Apatite Chromatography Support

Various apatite chromatography supports are available commercially, anyof which can be used in the practice of this invention. These includebut are not limited to hydroxyapatite and fluorapatite. “Ceramic”hydroxyapatite (CHUM) or “ceramic” fluorapatite (CF™) refer to forms ofthe respective minerals in which nanocrystals are aggregated intoparticles and fused at high temperature to create stable ceramicmicrospheres suitable for chromatography applications. Commercialexamples of ceramic hydroxyapatite include, but are not limited to CHTType I and CHT Type II. Commercial examples of fluorapatite include, butare not limited to CFT Type II. Unless specified, CHT and CFT refer toroughly spherical particles of any diameter, including but not limitedto 10, 20, 40, and 80 micron. HA Ultrogel™ refers to a productcomprising microfragments of non-ceramic hydroxyapatite embedded inporous agarose microspheres.

The choice of hydroxyapatite or fluorapatite, the type, and averageparticle diameter suitable for a particular application can bedetermined through experimentation by the skilled artisan.

The invention may be practiced in a packed bed column, afluidized/expanded bed column containing the hydroxyapatite orfluorapatite, and/or a batch operation where the hydroxyapatite orfluorapatite is mixed with the solution for a certain time.

Certain embodiments employ CHT or CFT packed in a column.

Certain embodiments employ CHT or CFT, packed in a column of about 5 mminternal diameter and a height of about 50 mm, for evaluating theeffects of various buffer conditions on the binding and elutioncharacteristics of a particular antibody preparation of antibodyfragment preparation.

Certain embodiments employ CHT or CFT, packed in columns of anydimensions required to support preparative applications. Column diametermay range from 1 cm to more than 1 meter, and column height may rangefrom 5 cm to more than 30 cm depending on the requirements of aparticular application.

Appropriate column dimensions can be determined by the skilled artisan.

2. Antibodies

Antibody preparations to which the invention can be applied may includeunpurified or partially purified antibodies from natural, synthetic, orrecombinant sources. Unpurified antibody preparations may come fromvarious sources including, but not limited to, plasma, serum, ascitesfluid, milk, plant extracts, bacterial lysates, yeast lysates, orconditioned cell culture media. Partially purified preparations may comefrom unpurified preparations that have been processed by at least onechromatography, precipitation, other fractionation step, or anycombination of the foregoing. The chromatography step or steps mayemploy any method, including but not limited to size exclusion,affinity, anion exchange, cation exchange, protein A affinity,hydrophobic interaction, immobilized metal affinity chromatography, ormixed-mode chromatography. The precipitation step or steps may includesalt or PEG precipitation, or precipitation with organic acids, organicbases, or other agents. Other fractionation steps may include but arenot limited to crystallization, liquid:liquid partitioning, or membranefiltration.

3. Antibody Fragments

Antibody fragment preparations to which the invention can be applied mayinclude unpurified or partially purified antibody fragments fromnatural, synthetic, or recombinant sources. Unpurified fragmentpreparations may come from various sources including, but not limitedto, plasma, serum, ascites fluid, milk, plant extracts, bacteriallysates, yeast lysates, or conditioned cell culture media. Antibodyfragment preparations may also include enzymatic digests of purified orpartially purified antibodies, such as but not limited to IgG monoclonalantibodies digested with plasmin, ficin, or pepsin. Partially purifiedpreparations may come from unpurified preparations that have beenprocessed by at least one chromatography, precipitation, otherfractionation step, or any combination of the foregoing. Thechromatography step or steps may employ any method, including but notlimited to size exclusion, affinity, anion exchange, cation exchange,protein A affinity, hydrophobic interaction, immobilized metal affinitychromatography, or mixed-mode chromatography. The precipitation step orsteps may include salt or PEG precipitation, or precipitation withorganic acids, organic bases, or other agents. Other fractionation stepsmay include but are not limited to crystallization, liquid:liquidpartitioning, or membrane filtration.

Description of the Method

In preparation for contacting the antibody preparation or antibodyfragment preparation with the hydroxyapatite or fluorapatite column, itis usually necessary to equilibrate the chemical environment inside thecolumn. This is accomplished by flowing an equilibration buffer throughthe column to establish the appropriate pH, conductivity, concentrationof salts; and/or the identity, molecular weight, and concentration ofnonionic organic polymer.

The equilibration buffer for applications conducted in bind-elute modemay include phosphate salts at a concentration of about 5-50 mM, orcalcium salts at a concentration of about 2-5 mM, but not mixtures ofphosphate and calcium. It may optionally include a nonionic organicpolymer at a concentration of about 0.01-50%, and a buffering compoundto confer adequate pH control. Buffering compounds may include but arenot limited to MES, HEPES, BICINE, imidazole, and Tris. The pH of theequilibration buffer for hydroxyapatite may range from about pH 6.5 topH 9.0. The pH of the equilibration buffer for fluorapatite may rangefrom about pH 5.0 to 9.0.

In one embodiment, the equilibration buffer contains sodium phosphate ata concentration of about 5 mM at a pH of 6.7, in the presence or absenceof MES or Hepes at a concentration of about 20-50 mM.

In one embodiment, the equilibration buffer contains a calcium salt at aconcentration of about 2.5 mM, in the presence of Hepes at aconcentration of about 20-50 mM and a pH of about 7.0.

The antibody preparation or antibody fragment preparation may also beequilibrated to conditions compatible with the column equilibrationbuffer before the invention is practiced. This consists of adjusting thepH, concentration of salts, and other compounds.

After the column and antibody preparation or antibody fragmentpreparation have been equilibrated, the antibody preparation may becontacted with the column. Said preparation may be applied at a linearflow velocity in the range of, but not limited to, about 50-600 cm/hr.Appropriate flow velocity can be determined by the skilled artisan.

In one embodiment of the bind-elute mode, a column equilibrated inphosphate to obtain a particular binding selectivity during columnloading may be switched to calcium to obtain a particular elutionselectivity. Or the opposite may be performed, with a columnequilibrated to calcium to obtain a particular binding selectivity, andthen switched to phosphate to obtain a particular elution selectivity.

In one embodiment of the flow-through mode, non-aggregated antibodyflows through the column and is collected, while aggregated antibodybinds to the column. The antibody preparation is followed with a washbuffer, usually of the same composition as the equilibration buffer.This displaces remaining non-aggregated antibody from the column so thatit can be collected. Retained aggregates may optionally be removed fromthe column with a cleaning buffer of about 500 mM sodium phosphate,among others.

In one embodiment of the flow-through mode, the desired antibodyfragment flows through the column and is collected, while contaminantsbind to the column. The fragment preparation is followed with a washbuffer, usually of the same composition as the equilibration buffer.This displaces remaining fragment from the column so that it can becollected. Retained contaminants may optionally be removed from thecolumn with a cleaning buffer of about 500 mM sodium phosphate, amongothers.

In one embodiment of an application conducted in bind-elute mode, somecombination of unwanted antibody fragments, intact non-aggregatedantibody, and aggregated antibody bind to the column. The antibodypreparation is followed with a wash buffer, usually of the samecomposition as the equilibration buffer. This removes unretainedcontaminants from the column. Unwanted antibody fragments may beselectively displaced by a wash buffer that removes fragments withoutremoving intact non-aggregated antibody. Intact non-aggregated antibodyis then eluted from the column under conditions that leave aggregatedantibody bound to the column. Retained aggregates may optionally beremoved from the column with a cleaning buffer of about 500 mM sodiumphosphate, among others.

In one embodiment of an application conducted in bind-elute mode, thedesired antibody fragment and contaminants bind to the column. Theantibody fragment preparation is followed with a wash buffer, usually ofthe same composition as the equilibration buffer. This removesunretained contaminants from the column. The desired antibody fragmentis then eluted from the column under conditions that leave contaminantsbound to the column. Retained contaminants may optionally be removedfrom the column with a cleaning buffer.

In one embodiment of the bind-elute mode, the wash buffer may have aformulation different than the equilibration buffer.

After use, the apatite column may optionally be cleaned, sanitized, andstored in an appropriate agent.

The invention may be practiced in combination with other purificationmethods to achieve the desired level of antibody or fragment purity. Theinvention may be practiced at any point in a sequence of 2 or morepurification methods.

It will be apparent to the person of ordinary skill that the inventionwill have a beneficial effect on removal of other contaminants, such asnucleic acids, endotoxin, virus, and complexes of antibody with leachedprotein A.

EXAMPLES

Considerable variation in chromatographic behavior is encountered fromone antibody or fragment preparation to another. This includes variationin the composition and proportion of non-antibody proteins, intactantibody, antibody fragments, and antibody aggregates that contaminatevarious preparations, as well as variation in the individual retentioncharacteristics of different constituents. This makes it necessary tocustomize the buffer conditions to apply the invention to its bestadvantage in each situation. This may involve adjustment of pH, theconcentration salts, the concentration of buffering components, and thecontent of nonionic organic polymer. Appropriate levels for the variousparameters and components can be determined systematically by a varietyof approaches. The following examples are offered for illustrativepurposes only.

Example 1 Dynamic Binding Capacity Comparison of Native andCalcium-Derivatized Hydroxyapatite

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. A sample of protein A purified IgG monoclonalantibody was applied to the column by in-line dilution at a proportionof 1 part antibody to 4 parts equilibration buffer. Dynamic breakthroughcapacity at 5% breakthrough was 114 mg/mL of hydroxyapatite. Theexperiment was repeated with an equilibration buffer of 20 mM Hepes, 3mM CaCl₂, 1 M NaCl, pH 6.7. Dynamic capacity at 5% breakthrough was 43mg/mL. The experiment was repeated with an equilibration buffer of 5 mMsodium phosphate, pH 6.7. Dynamic capacity at 5% breakthrough was 29mg/mL. The experiment was repeated with an equilibration buffer of 5 mMsodium phosphate, 1 M NaCl, pH 6.7. Dynamic capacity at 5% breakthroughwas 3 mg/mL. This example illustrates the dramatic improvement inantibody binding capacity that is achieved by calcium derivatizedapatite. It will be recognized by the skilled practitioner that asimilar benefit may be obtained by substituting magnesium for calcium.

Example 2 Purification of an IgG Monoclonal Antibody from Cell CultureSupernatant on Native Hydroxyapatite, Eluted with a Borate Gradient

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. A monoclonal antibody preparationconsisting of a mammalian cell culture supernatant previously filteredthrough a membrane with porosity of about 0.22 μm, and diafiltered toabout the same conditions as the equilibration buffer was applied to thecolumn. The column was eluted with a linear gradient to 1 M sodiumborate, 5 mM sodium phosphate, pH 7.0. The majority of contaminatingproteins eluted before the antibody. Non-aggregated antibody eluted atan average conductivity of about 5 mS/cm. Aggregates eluted later. Thecolumn was cleaned with 500 mM sodium phosphate, pH 7.0. It will berecognized by the person of ordinary skill in the art that elutedantibody may be further purified by additional purification methods, andthat the low conductivity and buffer capacity of the eluted antibodyfraction will facilitate such methods.

Example 3 Purification of an IgG Monoclonal Antibody from Cell CultureSupernatant on Native Hydroxyapatite, Eluted with a Monocarboxylic Acid(Lactate) Gradient

A column of hydroxyapatite, CHT Type I, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 600 cm/hr with 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. 100 microliters of a monoclonalantibody preparation consisting of a mammalian cell culture supernatantpreviously filtered through a membrane with porosity of about 0.22 μm,was injected onto the column and the column washed with 2 column volumesof equilibration buffer. The column was eluted with a 20 column volumelinear gradient to 1 M sodium lactate, 20 mM Hepes, pH 7.0. The majorityof contaminating proteins eluted before the antibody and most of theremainder eluted later. Non-aggregated antibody eluted at an averageconductivity of about 20 mS/cm. Aggregates eluted later. The column wascleaned with 500 mM sodium phosphate, pH 7.0.

Example 4 Purification of an IgG Monoclonal Antibody from Cell CultureSupernatant on Native Hydroxyapatite, Eluted with a Borate Gradient

The same column was prepared with the same buffers but with a differentIgG monoclonal antibody. The majority of contaminating proteins elutedas previously but the antibody eluted only partially within thegradient. The run was repeated but with 20 mM phosphate in theequilibration and elution buffers. Under these conditions, the antibodyeluted completely within the gradient. Antibody aggregate eluted afternon-aggregated antibody. This example illustrates one way to adapt theprocedure to antibodies that may not elute fully within the gradient.The phosphate concentration may be increased more if necessary.Alternatively or additionally, the borate concentration and/or pH of theeluting buffer may be increased.

Example 5 Purification of an IgG Monoclonal Antibody from Cell CultureSupernatant on Calcium Derivatized Hydroxyapatite, Eluted with a BorateGradient

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 2.5 mMcalcium chloride, 20 mM Hepes, pH 7.0. A monoclonal antibody preparationconsisting of cell culture supernatant previously filtered through amembrane with porosity of about 0.22 μm and diafiltered to about thesame conditions as the equilibration buffer was applied to the column.The column was eluted with a linear gradient to 1 M sodium borate, 2.5mM calcium chloride, 10% PEG-600, pH 7.0. The majority of contaminatingproteins eluted before the antibody. Antibody aggregate eluted afternon-aggregated antibody. The column was cleaned with 500 mM sodiumphosphate, pH 7.0. PEG is known to have the general effect of enhancingthe separation between fragments, intact antibody, and aggregates onhydroxyapatite. The skilled practitioner will recognize how to adjustthe PEG concentration to optimize the results.

Example 6 Monoclonal Antibody Capture on Calcium-DerivatizedHydroxyapatite and Elution in a Sulfate Gradient

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. Cell culture supernatant containingapproximately 60 mg monoclonal IgG was equilibrated to 5 mM calcium byaddition of 1 M calcium chloride at a proportion of 0.5%, then filteredto 0.22 microns. The sample was applied to the column. No antibody wasdetected in the flow-through. The column was washed with equilibrationbuffer, then eluted with a 20 column volume (CV) linear gradient to 20mM Hepes, 3 mM CaCl₂, 0.5 M sodium sulfate, pH 6.7. The antibody elutedin a single peak at about 0.25 M sodium sulfate.

Example 7 Monoclonal Antibody Capture on Calcium-DerivatizedHydroxyapatite, Conversion to Native Hydroxyapatite, and Elution in aPhosphate Gradient

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. Cell culture supernatant containingmonoclonal approximately 40 mg IgG was equilibrated to 5 mM calcium byaddition of 1 M calcium chloride at a proportion of 0.5%, then filteredto 0.22 microns. The sample was applied to the column. No antibody wasdetected in the flow-through. The column was washed with 5 mM sodiumphosphate, 20 mM MES, pH 6.7, then eluted with a 20 CV linear gradientto 300 mM phosphate, pH 6.7. The antibody eluted in a single peak atabout 165 mM sodium phosphate. This example illustrates the use ofcalcium-derivatized hydroxyapatite to obtain high binding capacity,followed by conversion to and elution from native hydroxyapatite.

Example 8 Intermediate Purification of a Monoclonal Antibody by Bindingin the Presence of Calcium, Conversion to Native Apatite, and Elution ina Sodium Chloride Gradient

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. Approximately 50 mg of protein A purifiedmonoclonal IgG was equilibrated to 5 mM calcium by addition of 1 Mcalcium chloride at a proportion of 0.5%, then filtered to 0.22 microns.The sample was applied to the column. No antibody was detected in theflow-through. The column was washed with 20 mM Hepes, 10 mM sodiumphosphate, pH 6.7, then eluted with a 20 CV linear gradient to 20 mMHepes, 10 mM phosphate, 1 M sodium chloride, pH 6.7. The antibody elutedin a single peak at 0.6 M sodium chloride, followed by a well-separatedaggregate peak.

Example 9 Unwanted Fragment and Aggregate Removal from a PartiallyPurified IgG Monoclonal Antibody, on Native Hydroxyapatite, Eluted witha Borate Gradient

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. A monoclonal antibody preparationpreviously purified by protein A affinity chromatography was applied tothe column. The column was eluted with a linear gradient to 1 M sodiumborate, 5 mM sodium phosphate, 20 mM Hepes, pH 7.0. The majority offragments eluted before the antibody. Antibody aggregates and othercontaminating proteins eluted after non-aggregated antibody. The columnwas cleaned with 500 mM sodium phosphate, pH 7.0.

Example 10 Bind-Elute Mode, Comparison of Monoclonal IgM Elution inPhosphate and Sulfate Gradients

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 200 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. Cell supernatant containing a monoclonal IgMantibody was applied to the column. The column was eluted with a 20 CVlinear gradient to 20 mM Hepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH6.7. The center of the IgM peak eluted about 415 mM sodium sulfate. DNAeluted at 855 mM sulfate under these conditions. IgM aggregates did notelute within the sulfate gradient and were removed in a subsequent washstep with 500 mM phosphate. The experiment was repeated except that thecolumn was equilibrated with 10 mM sodium phosphate pH 6.7 and elutedwith a 20 CV linear gradient to 500 mM sodium phosphate, pH 6.7. Thecenter of the IgM peak eluted at about 207 mM phosphate, essentiallyco-eluting with DNA as revealed by its elution at 205 mM phosphate. IgMaggregates were only partially eliminated. This example againillustrates the dramatic difference of selectivity between sulfate andphosphate gradients, specifically and dramatically highlights howsulfate gradients are more effective for removal of DNA from IgMpreparations, and specifically illustrates the superior ability ofsulfate gradients to eliminate aggregates.

Example 11 Purification of Fab

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. A Fab preparation from papaindigestion of an IgG monoclonal antibody was applied to the column. Thecolumn was eluted with a linear gradient to 1 M sodium borate, 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. The majority of contaminating Fcfragments eluted before the Fab. Intact antibody eluted after the Fab.The column was cleaned with 500 mM sodium phosphate, pH 7.0.

Example 12 Purification of F(ab′)₂

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 5 mMsodium phosphate, 20 mM Hepes, pH 7.0. A F(ab′)₂ preparation from pepsindigestion of an IgG monoclonal antibody was applied to the column. Thepreparation contained no intact IgG. The column was eluted with a lineargradient to 1 M boric acid, 5 mM sodium phosphate, pH 7.0. The majorityof contaminating Fc fragments eluted before the F(ab′)₂. The column wascleaned with 500 mM sodium phosphate, pH 7.0.

Example 13 Purification of F(ab′)₂

A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 2.5 mMcalcium chloride, 20 mM Hepes, pH 7.0. A F(ab′)₂ preparation from pepsindigestion of an IgG monoclonal antibody was applied to the column. Thecolumn was eluted with a linear gradient to 1 M boric acid, 2.5 mMcalcium chloride, 20 mM Hepes, pH 7.0. The majority of contaminating Fcfragments eluted before the F(ab′)₂. The preparation contained no intactIgG. The column was cleaned with 500 mM sodium phosphate, pH 7.0.

Example 14 2-Step Fab Purification on Calcium-Derivatized Hydroxyapatiteand Cation Exchange Chromatography

A column of hydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 50 mmheight, was equilibrated with 10 mM sodium Hepes, 2.5 mM calciumchloride, pH 7.0 at a linear flow rate of 300 cm/hr. Calcium chloridewas added to a Fab digest to a final concentration of 2.5 mM, thenloaded onto the column. The Fab was unretained and flowed through thecolumn at a purity of about 95%. The pH of the Fab fraction was titratedto pH 5.5 by addition of 1 M MES, pH 5.5, and applied to a cationexchange column (BIA Separations, CIM S) equilibrated to 20 mM MES, pH5.5. The Fab bound and was eluted in a gradient of increasing pH,yielding a Fab fraction of purity greater than 99%. This exampleillustrates 2 important points: 1) the unique selectivity of calciumderivatized apatite, and 2) its ability in this case to elute theproduct at low conductivity and buffer capacity so as to facilitate asubsequent cation exchange chromatography step. It will be apparent tothe skilled practitioner that the order of steps could be reversed toachieve a similar result, and that an additional step or steps could beadded to achieve clinical purity.

Example 15 2-Step Fab Purification on Calcium-Derivatized Hydroxyapatiteand Hydrophobic Interaction Chromatography

A column of hydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 50 mmheight, was equilibrated with 10 mM sodium Hepes, 2.5 mM calciumchloride, 1 M sodium chloride, pH 7.0 at a linear flow rate of 300cm/hr. A Fab preparation was titrated to 2 M sodium chloride, 2.5 mMcalcium chloride, pH 7.0, then loaded onto the hydroxyapatite column.The Fab was unretained and flowed through the column at a purity ofabout 95%. The Fab fraction was diluted 1:1 with 4 M sodium chloride, 20mM Hepes, pH 7.0, then loaded onto a hydrophobic interaction column(Tosoh Phenyl 600M) equilibrated to 2 M sodium chloride, 5 mM EDTA, 20mM Hepes, pH 7.0. The Fab bound and was eluted in a gradient ofdecreasing salt concentration, yielding a Fab fraction of purity greaterthan 99%. The experiment was repeated, with the original Fab preparationdiluted in 9 parts spent mammalian cell culture supernatant. The resultswere essentially the same, demonstrating that the majority of host cellproteins, as well as Fc-containing proteins, bound to the calciumderivatized hydroxyapatite. These experiments illustrate severalimportant points: 1) the demonstrate another aspect of the uniqueselectivity of calcium-derivatized apatite, in this case illustratingits ability to bind contaminants even at high conductivity, 2) how thesample application conditions facilitate application of the HA-elutedFab to a hydrophobic interaction chromatography column, and 3) that themethod works equally well with simple preparations, such as enzymedigests, or with complex preparations, such as cell culturesupernatants. It will be apparent to the skilled practitioner that theorder of steps could be reversed to achieve a similar result, and thatan additional step or steps could be added to achieve clinical purity.

Example 16 2-Step Fab Elution from Native Hydroxyapatite by Conversionto Calcium Derivatized Hydroxyapatite, and Cation ExchangeChromatography

A column of hydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 50 mmheight, was equilibrated with 5 mM sodium phosphate, 10 mM Hepes, pH 7.0at a linear flow rate of 300 cm/hr. A Fab preparation was titrated to 5mM phosphate and loaded onto the hydroxyapatite column. The Fab wasretained and eluted with a step to 10 mM Hepes, 2.5 mM calcium chloride,pH 7.0. Purity was greater than 95%. The pH of the Fab fraction wastitrated to pH 5.5 by addition of 1 M MES, pH 5.5, and applied to acation exchange column (BIA Separations, CIM S) equilibrated to 20 mMMES, pH 5.5. The Fab bound. The column washed with 10 mM MES, 2.5 mMEDTA, pH 5.5, then eluted in a linear pH gradient to 10 mM Hepes, pH7.5, yielding a Fab fraction of purity greater than 99%. This exampleillustrates an important benefit of switching from native hydroxyapatitefor sample loading to calcium-derivatized apatite for elution: Theability of Fab to bind native apatite under these conditions provides ameans to concentrate the product from a dilute feed stream. The skilledpractitioner will recognize that the cation exchange step couldoptionally have been eluted with a gradient of increasing conductivity,or a combination of pH and conductivity.

Example 17 1-Step Purification of F(ab′)₂ on Native Hydroxyapatite withan Increasing Sodium Chloride Gradient

F(ab′)₂ was prepared from purified IgG by enzymatic digestion withpepsin. It was applied to a column of native hydroxyapatite in 5 mMsodium phosphate and eluted with a gradient to 5 mM phosphate, 500 mMsodium chloride, pH 7. F(ab′)₂ eluted at about 250 mM sodium chloride.Most of the Fc contamination eluted after the F(ab′)₂ peak, but someco-eluted with the product. This contamination was mostly avoided insubsequent experiments by performing a wash, after sample injection, of25 mM sodium phosphate, pH 7.0. After the wash, the column wasre-equilibrated to 5 mM phosphate and eluted with a sodium chloridegradient as described above. The column was subsequently cleaned with500 mM phosphate, pH 7.0.

Example 18 1-Step Purification of F(ab′)₂ on Native Hydroxyapatite withan Increasing Sodium Borate Gradient

F(ab′)₂ was prepared from purified IgG by enzymatic digestion withpepsin. It was applied to a column of native hydroxyapatite in 5 mMsodium phosphate, 20 mM Hepes, pH 7.0, and eluted with a gradient to 5mM phosphate, 500 mM sodium borate, pH 7. Most of the Fc contaminationeluted after the F(ab′)₂ peak but some co-eluted. This contamination wasmostly avoided in subsequent experiments by performing a wash, aftersample injection, of 25 mM sodium phosphate, pH 7.0. After the wash, thecolumn was re-equilibrated to 5 mM phosphate and eluted with a sodiumchloride gradient as described above. It will be recognized by theskilled practitioner that the low conductivity and buffering capacity ofthe borate-eluted F(ab′)₂ make it better suited for subsequentpurification by cation exchange chromatography than elution in a sodiumchloride gradient. It will be equally recognized that the substitutionof borate with monocarboxylic acids or zwitterions with molarconductivities lower than sodium chloride may confer a similar benefit.

Example 19 Purification of F(ab′)₂ on Calcium Derivatized Hydroxyapatitewith an Increasing Ammonium Sulfate Gradient

F(ab′)₂ was prepared from purified IgG by enzymatic digestion withpepsin. It was applied to a column of calcium derivatized hydroxyapatitein 2.5 mM calcium chloride, 20 mM Hepes, pH 7.0. A small amount ofnon-F(ab′)₂ proteins were unretained and flowed through the column. Thecolumn was washed with equilibration buffer, then eluted with a lineargradient to 1.0 M ammonium sulfate, 2.5 mM calcium chloride, 20 M Hepes,pH 7.0. The skilled practitioner will recognize that elution in ammoniumsulfate facilitates application of the F(ab′)₂ fraction to a hydrophobicinteraction column for additional purification.

Example 20 Bind-Elute Mode, Comparison of DNA Elution in Phosphate andSulfate Gradients

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. A sample of DNA isolated from salmon spermwas applied to the column. The column was eluted with a 20 CV lineargradient to 20 mM Hepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH 6.7. Thecenter of the DNA peak eluted at about 855 mM sodium sulfate. Theexperiment was repeated except that the column was equilibrated with 10mM sodium phosphate pH 6.7 and eluted with a 20 CV linear gradient to500 mM sodium phosphate, pH 6.7. The center of the DNA peak eluted atabout 205 mM sodium phosphate. This example illustrates the dramaticdifference between selectivity of sulfate and phosphate gradients. Itwill be apparent to the skilled practitioner that these results alsoshow the ability of sulfate gradients to achieve more effective removalof DNA than phosphate gradients.

Example 21 Bind-Elute Mode, Comparison of Endotoxin Elution in Phosphateand Sulfate Gradients

A column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 20 mMHepes, 3 mM CaCl₂, pH 6.7. A sample of endotoxin prepared by phenolextraction from Salmonella enterica serotype typhimurium was applied tothe column. The column was eluted with a 20 column volume (CV) lineargradient to 20 mM Hepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH 6.7. Aminor fraction of endotoxin eluted early in the gradient, followed by aDNA contaminant peak at 855 mM sodium sulfate. The majority of theendotoxin failed to elute and was removed from the column by cleaning itwith 500 mM sodium phosphate, pH 6.7. The experiment was repeated exceptthat the column was equilibrated with 10 mM sodium phosphate pH 6.7 andeluted with a 20 CV linear gradient to 500 mM sodium phosphate, pH 6.7.A minor fraction of the endotoxin, corresponding to the early elutingpopulation in the sulfate gradient, failed to bind in phosphate andflowed through the column immediately upon application. The center ofthe primary endotoxin peak eluted at 85 mM sodium phosphate. Thisexample illustrates the dramatic difference between selectivity ofsulfate and phosphate gradients in general, specifically illustrates theability of sulfate gradients to achieve unique separations amongdifferentially phosphorylated biomolecules, and specifically illustratesthat some phosphorylated biomolecules do not elute from at least someapatite chromatography supports in sulfate gradients conducted in theabsence of phosphate. It will be apparent to the skilled practitionerthat these results also show the ability of sulfate gradients to achievemore effective removal of endotoxin than phosphate gradients.

Example 22 Improved pH Control by the Application of Borate

A column of hydroxyapatite was equilibrated to 5 mM sodium phosphate, pH7.0. A gradient step of 0.5 M sodium chloride, 5 mM sodium phosphate, pH7.0 was applied to the column. This caused the pH to drop to about pH5.9. The column was re-equilibrated to 5 mM phosphate pH 7.0. A gradientstep of 0.5 mM sodium chloride, 5 mM sodium phosphate, 50 mM sodiumborate, pH 7.0 was applied to the column. Column pH dropped only to pH6.7. It will be understood by the skilled practitioner that the sameapproach can be used to control pH in any situation where theintroduction of an eluting agent causes an unacceptable reduction of pH,and that the borate concentration can be adjusted to achieve the desireddegree of pH control. Like borate, the application of lactate to anequilibrated apatite support causes an increase in pH, which canlikewise be exploited to manage uncontrolled pH reduction caused bychlorides. The skilled practitioner will recognize that othermonocarboxylic acids or zwitterions may be substituted to produce asimilar effect.

It will be understood by the person of ordinary skill in the art how tooptimize and scale up the results from experiments such as thosedescribed in the above examples. It will also be understood by suchpersons that other approaches to method development, such as but notlimited to high-throughput robotic systems, can be employed to determinethe conditions that most effectively embody the invention for aparticular antibody.

Additional Optional Steps

The present invention may be combined with other purification methods toachieve higher levels of purification, if necessary. Examples include,but are not limited to, other methods commonly used for purification ofantibodies, such as size exclusion chromatography, protein A and otherforms of affinity chromatography, anion exchange chromatography, cationexchange chromatography, hydrophobic interaction chromatography,immobilized metal affinity chromatography, mixed mode chromatography,precipitation, crystallization, liquid:liquid partitioning, and variousfiltration methods. It is within the purview of one of ordinary skill inthe art to develop appropriate conditions for the various methods andintegrate them with the invention herein to achieve the necessarypurification of a particular antibody.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, chromatographyconditions, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired performance sought to beobtained by the present invention.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

1. A method for purifying at least one non-aggregated antibody or atleast one F(ab′)₂ antibody fragment from an impure preparationcontaining said antibody or antibody fragment comprising: (a) contactingthe impure preparation with an apatite chromatography support, whereinthe apatite chromatography support is in a calcium-derivatized form whenit is contacted with the impure preparation containing said antibody orantibody fragment and wherein the antibody or antibody fragment binds tothe calcium-derivatized form of the apatite chromatography support; and(b) substantially converting the calcium-derivatized apatitechromatography support to the support's native form, thereby forming anative form apatite chromatography support; and subsequently (c) elutingthe antibody or antibody fragment.
 2. The method of claim 1, wherein thenative form apatite chromatography support is eluted with phosphate asthe primary eluting ion.
 3. The method of claim 1, wherein the impurepreparation contains the antibody and the antibody is eluted in theeluting step.
 4. The method of claim 1, wherein the impure preparationcontains the antibody fragment and the antibody fragment is eluted inthe eluting step.